Increased stability approach

László Cseresznyés Ada Yazan

Beatriz Mesquita Taline Frantz

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Link to the docs page: https://docs.google.com/document/d/16ZzK7eDNBL6F3MkKbrmYwtSyVOBJzyO09PpXXR_nnpA/edit?tab=t.0

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Academic Background

The bacteriophage MS2 exhibits a net negative surface charge under natural aqueous conditions, which generates electrostatic repulsion between virus particles, preventing aggregation even in high ionic strength environments. This electrostatic repulsion is a critical defense against destabilizing forces that might cause the phage to aggregate. (Wong et al., 2009) With these insights, strategies for enhancing MS2 stability must be focusing on preserving electrostatic repulsion and preventing aggregation.

1. Concept

We aim to create a lysis protein (L) of the MS2 bacteriophage to enhance its stability and functionality, especially when it needs to overcome potential resistance mechanisms in E. coli. By using the protein engineering techniques we learnt throughout the course, we want to use in silico methods to create a more robust protein that would be less dependent on bacterial chaperones and be more resistant to degradation as well. Also, while assessing the properties of our engineered lysis.

So, the ultimate goal is to improve the efficacy of MS2 phages as a therapeutic tool in phage therapy, providing a potential solution to the growing problem of antibiotic resistance. Increasing the stability of a protein can rely on:

Creating mutations that enhance the stability of the lysis protein, ensuring it remains functional under a wide range of conditions.

It could work to trigger mutations to change the stability of the protein in silico, then compare the protein structure and its folding.

Methods to Be Used

We've chosen many advanced techniques to improve the functionality and stability of the MS2 bacteriophage lysis protein to use in our project. In silico mutagenesis will be used to design mutations on the hydrophobic cores of the protein which could improve stability. We think the consideration of these modifications is structured advances or changes to the structure and stability of the protein by evaluating the yield of experiments through structure propotionate models and AlphaFold. MSA and ESM colab will be helpful in the identification of conserved residues, so the mutations are added within the natural evolutionary tree.

**Hypotheses

1.** Based on the read articles, we obtained the information that disulfide bonds are able to help stabilize protein structures but may result in increased reactivity depending on some parameters.

Karimi, M., Ignasiak, M., Chan, B. et al. Reactivity of disulfide bonds is markedly affected by structure and environment: implications for protein modification and stability. Sci Rep 6, 38572 (2016). https://doi.org/10.1038/srep38572

As we were thinking, however it may be good to try in silico mutagenesis techniques and try to fold the obtained mutated protein structure; it would be good to rely on disulfide bonds on stabilizing the protein as we are conducting mutagenesis (in silico).

• This way, maybe we can try and have more aggressive mutations to later stabilize using disulfide bonds?

2. Background study

To understand how L-protein is folded by the DnaJ shaperon, we examined the structure of L-protein. It consists of a low-confirmational flexible tale part, and a high-confirmational alpha-helix. Residues 1-40 make up the N-terminal domain (the tail and the first third of the alpha-helix), while the last two-third of the alpha-helix is the transmembrane part.

Questions:

• does the binding site (1-40 residues) mean that this is the aggregation-prone region, which is too unstable to fold on its own, therefore the mutation should be on this regoin?
• DnaJ protein has a high affinity to bind to exposed hydrophobic patches. Does this mean that these exposed hydrophobic patches are the problem for the L-protein to stably fold on its own, so mutations should target these patches?

L-protein 3D structure using alphafold2_ptm.